CA2090677C

CA2090677C - Method for culturing t lineage precursor cells

Info

Publication number: CA2090677C
Application number: CA002090677A
Authority: CA
Inventors: Yoshihiro Watanabe
Original assignee: Japan Tobacco Inc
Current assignee: Japan Tobacco Inc
Priority date: 1991-07-04
Filing date: 1992-07-01
Publication date: 1996-08-27
Anticipated expiration: 2012-07-01
Also published as: DE69229333D1; EP0552372A1; KR930702508A; EP0552372A4; WO1993001277A1; EP0552372B1; US5792655A; KR0152100B1; ATE180830T1; US5476780A; CA2090677A1

Abstract

The present invention relates to a method for culturing lineage precursor cells to produce matured T cells.
This method comprises a step of culturing the T lineage precursor cells or tissue containing T cells in a nutrient medium contacting a gas phase having a oxygen content ranging from 40% to 95%, to obtain matured T cells in terms of their phenotypes of differentiation antigens and of their functions.
Then, this method allows, by a submerged (suspension) culture which is a general method of culturing cells or tissues, to easily and efficiently produce matured T cells.

Description

~ 2090677 "METI{OD E OR CULTURING T LI~EAGE ~RECURSOR CELLS"
T~chnical ~ield The present invention relates to a method f or culturing T lineage precursor cells, which enables derivation and generation of matured T cells differentiated from the T
lineage precursor cells. The invention further relates to T
cells obtained by using this method.
10 ~ackground Art The earliest of T cells exist mainly in bone marrow or fetal liver, as a form of lymphoid 6tem cells differentiated from hematopoietic stem cells having an ability to differentiate into multilineage cells. These earliest stem cells emigrate to the thymuæ and mature into T cells through a series of differentiation stages. The differentiation and maturation of the T precursor cells are greatly under the influence of thymic stromal cells. After T precursor cells enter into the thymic cortex, the T precur60r cells 20 differentiate and mature into T cells through rapid cell division and proliferation under the control of adhesion witll 6tromal cells comprising mainly thymic epithelial cells which participate in differentiation and maturation of tlle T
precursor cell6, adhesion witll myeloid cells such as macrophage6, and humeral factors produced therefrom. After undergoing several differentiation stages thymocytes flow out to the bloodstream from the cortico-medullary junction of thymus, and emigrate to peripheral lymphoid organs. The emigrated thymocyte6, i.e. T cells, further dif~erentiate and 30 mature therein.
To study the detailed process of differentiation and maturation of the T precursor cells, a culture system capable of achieving in-vitro proliferation, differentiation, and maturation of T ceIls is required.
On the other hand, it is considered that abnormality of differentiation or of functions of T cells due to s~v I ~
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~ 2~90677 ,; 7~tion with infectious bacteria and/or a foreign antigen is one great cause of allergic disease6 or various autoimmune diseases. (e.g., Animesh A. et al, Science, 248, 1380-1388, 1990; Irun R. Cohen et al, Immunology Today, 12, No. 4, 105-110, lg91; and Lars E~lareskog et al, Immunological Reviews, No. 118, 285-310, 1990).
At present, as therapeutic drugs for such diseases, non-specific anti-inflammatory agents and immunosuppressants of T cell functlons are u6ed as suppressors. However, 10 essential cure of the diseases has not been achieved due to their non-6pecificity.
To overcome these problems, extensive studies on vaccine therapy and serum vaccine therapy ~lave been made, comprising respectively (l)specifying an antigen which causes disease and applying the antigen, and ( 2 ) specifying said antigen and applying suppressive factors specific for the diseases which are represented by antibodies against the antigen. A cell vaccine therapy using T cells whose antigen specificities has been proved is considered to be an effective 20 therapeutic method for diseases which is assumed to be caused by abnormal differentiation and functional abnormality of T
cells (e.g., Jose C. Gutierre_-Ramos et al, Nature, 346, 271-274, 1990 and Kohei Ota et al, Nature, 346, 183-187, 1990). However, methods capable of deriving T cells having appropriate antigen specificity in vitro are limited to a particular method.
In fact, although a submerged (suspension) culture in which cells or tissue6 are submerged or suspended in a nutrient medium is well known as a method generally used for 30 culture of cells or tissues, it i8 impossible to differentiate and mature T precursor cells into T cells in accordance with ~uch a conventional method.
At present, as tlle sole culture method capable of achieving differentiation and maturation of the T precursor cells, only a so-called "afloat culture" is known, in which a fetal thymus is cultured on an interface between the gas -~ 2~90~77 phase and the nutrient medium under atmospheric air whose carbon dioxide concentration is controlled to 5% (e.g.~
Jenkinsen E.J. et al, Eur. J. Immunology, 12, 583, 1982 and Osamu Mazda et al, J. ~xp. Med., 173, 539-547, 1991). ~lowever, this method requires special tools or apparatuses, and dif-ferentiation of cells on the liquid surface side is insufficient. In addition to these drawbacks, this method is not suitable for the culture of the T precursor cells accompanying mass proliferation of cells. Therefore, it is 10 difficult to perform a large scale culture.
Strong demand has, therefore, arisen for developing a culture method capable of realizing and achieving derivation of matured T cells differentiated from T precursor cells in terms of their phenotypes of differentiation antigens and of their functions, by using the submerged Isuspension) culture as a general method of culturing cells or tissues.
International Patent Application Published under serial number Wo89/11530 on November 30, 1989 (Buser et al. ) discloses a method for proliferating a human B blastic cell 20 line by raising the dissolved oxygen concentration in the culture medium by steadily supplying a desired amount of oxygen into the medium placed in a sealed vessel, through a silicon pipe connected to a gas bomb sealed with a gas mixture containing a desired amount of oxygen. In said culture according to the method, the proliferation of the human B
blastic cell line is found but differentiation and maturation of the cells is not achieved. The present invention is capable of achieving proliferation of T precursor ceIls, and of di$ferentiation and maturation of them in terms of their 30 phenotypes of differentiation antigens and of their functions, by using a simpler culture method than that disclosed in above application under a higher concentration of dissolved oxygen in the medium.
Di~clo~ure of Invention It is an object of the present invention to provide e ~ 2090~77 a culture method capable of realizing and achieving derivation of matured T cells, differentiated from T precursor cells in terms of their phenotypes of differentiation antigens and of their functions, by u6ing the submerged (suspension) culture as a general met~lod of culturing cells or tissues.
In order to achieve the object, the present inventors made extensive studies on a method of in-vi tro culture of T lineage precursor cells and found that matured T cells, differentiated from T lineage precursor cells in lo terms of t}leir phenotypes of differentiation antigens or their functions, could very be efficiently and easily derived and generated by culturing T lineage precursor cells under a condition wherein t~le dissolved oxygen concentration in a nutrient medium was higher than that in the normal atmospheric air , i . e ., under a condition of high oxygen concentration , thereby achieving the present invention.
That is, a method of culturing T lineage precursor cells according to the present invention comprises a step of culturing the T lineage precursor cells in a nutrient medium 20 under conditions such that the dissolved oxygerl concentration in the nutrient medium is higher than that present in the normal atmospheric air.
According to the present invention, by using a submerged (suspension) culture well known a6 a general method of culturing cells or tissues, matured T cells, normally dif ferentiated from T precursor cells in terms of their phenotypes of differentiation antigens or their functions, can be derived and the T precursor cells can be proliferated. In addition, according to the present invention, the culture can 30 be very easily performed without using special tools or apparatuses, and a large scale culture can be performed.
The present invention is described in detail below.
The terms "T lineage precursor cells", "T precursor cells", "tissue(s) containing T precursor cells", "inter-stitial cells of lymphoid tissues", "nutrient medium", "dissolved oxygen concentration in nutrient medium", and i..
~5 , `` ~ 2090677 "matured T cells differentiated in terms of t~leir phenotypes of differentiation antigens or their functions" are defined as follows:
a) "T lineage Precursor cells"
The "T lineage precurfior cells" used in the present invention mean "T precursor cells", "tissue(s) containing T
precursor cells", or a "combination of the T precursor cells and the interstitial cells of lymphoid tissues". The term "T
lineage precursor cells" can include hematopoietic stem cells.
lo The "T lineage precursor cells" can be collected from any tissue(s~ having T precursor cells regardless of the species, sex, and age of the doner. Preferably, the T lineage precursor cells can be collected from a mammal such as a rat, a mouse, a rabbit, a horse, or a cow. More preferably, T
lineage precursor cells are collected from a fetus or neonate of said mammal, and can be collected from a human.
A population of cells participating in the immune response is generically called immunocompetent cells. Among the i no~ Lent cells, T- and s-lineage lymphoid cells are 20 related to antigen memory and play principal roles of immune responses. The present invention relates to the T-lineage cells. The origins of the T-lineage cells are lymphoid stem cells differentiated from hematopoietic stem cells existing mainly in bone marrow or fetal liver, and the lymphoid stem cells develop into T lineage stem cells through differen-tiation. The T lineage stem cells emigrate to the thymus and mature to T cells having an ability of playing a role of cellular immunity through respective stages of differentiation accompanying proliferation.
The term "T precursor cells" used in the present invention means any T lineage cells at any differentiation stage in a series of differentiation and maturation processes f rom lymphoid stem cells to matured T cells, as described above. For example, the population of "fetal thymocyte(s)"
referred to in this specification contains a large number of T lineage stem cells which have emigrated to thymus, and is . S

~ 2090677 interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-ll, interleukin-12, TNF (Tumour Necrosis Factor), y-interferon, a granulocyte macrophage colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating factor (G-CSF), or a macrophage colony-stimulating factor (M-CSF), depending on application purposes. By using nutrient medium containing an antigen which induces infectious diseases due to such as a hepatitis virus, an HIV (Human Immunodeficiency Virus) and so on, allergic 10 diseases and autoimmune diseases, T cell vaccines for suppressing and treating t~e symptoms of infectious diseases, inflammation, allergy, and autoimmune di6ea~es induced by t~le antigens can ~e derived and generated.
c) "Dissolved Oxygen Concentration in Nutrient Medium"
The term "dissolved oxygen concentration in nutrient medium" used in the present invention means the concentration of oxygen dissolved in a nutrient medium (liquid phase) for culturing the T lineage precursor cells defined in a ) . The 20 p~lrase "dissolved oxygen concentration in nutrient medium under normal atmospheric air" indicates the dissolved oxygen concentration in a nutrient medium saturated with natural atmospheric air which is not artificially modified. Therefore, for example, the dissolved oxygen concentration in a nutrient medium under the condition that the total pressure of the gas phase contacting the nutrient medium is 1 atm, that the oxygen partial pressure of the gas phase is 0 . 4 atm, and that the temperature is 37C" indicates that said dissolved oxygen concentration is twice of that of normal atmospheric air, 30 namely, l atm atmospheric air, in accordance with Henry's law which says that the solubility of gas at constant temperature is proportional to its partial pressure as far as the pressure is not so high.
As a means for raising the dissolved oxygen concen-tration in the medium, any means can be used if the means has a capacity for supplying oxygen to raise the amount of oxygen ~, ~., ~L

~ 2090677 diE~olving in the nutrient medium. In particular, it is preferable to 6ubstantially raise the dissolved oxygen concentration in the nutrient medium (liquid phase) by raising the oxygen ~u~ Llcltion in the gas phase contacting with the nutrient medium (liquid phase) within the range ûf 40 vol%
thrûugh 95 vol%, preferably 60 vol% through 95 vol%.
The oxygen concentration is expressed in different units such as 96, atm (atmûsphere), mmHg, and M (mol) herein.
Any unit of the oxygen concentrations expressed can be 10 converted into any other. The oxygen concentration in the present invention need not be expressed in a specif ic unit, as a matter ûf course.
The solubility of oxygen (which can be expressed in unit such as M (mol) ) is proportional to the partial pressure of oxygen in the gas phase (which can be expressed in unit ~;uch as vol % ) . Then by raising the oxygen concentration in the gas phase contacting with the nutrient medium ( liquid phase) to 40 vol% - 95 vol% and preferably to 60 vol96 - 95 vol%, which are preffered embodiments of the present 20 invention, the di6solved oxygen concentration in the nutrient medium can be raised to approximately 360 ,uM - 855 ,LM and to approximately 540 ,uM - 855 ~M, respectively.
It is also easily understood for the skilled in the art that the dissolved oxygen concentration in the medium can be raised by slightly elevating the pressure of the gas phase even if the oxygen concentration of the gas phase is 60 vol%
or less, or 40 vol% or less.
In case of necessity of replacing a part of the medium during the culture, it i8 preferable to control, in 30 advance, the dissolved oxygen concentration in a medium for replacement to the same dissolved oxygen concentration in the former medium. Alternatively, the dissolved oxygen concentration in the medium for replacement can be controlled by using air flow containing a high amount of oxygen immediately after the replacement.

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~ 209~677 d) "Matured T cells Differentiated in Terms of their Phenotypes of Differentiation Antigens or their Functions"
T lineage stem cells differentiated and developed from lymphoid stem cells derived from hematopoietic stem cells proliferate and mature through each stage of differentiation and maturation under the control of adhesion with the thymic interstitial cells and of ~n<log.on-~us humoral factors.
Each stage of differentiation and maturation can be 8a tB
.

~ 2~90677 d) "Matured T cells Differentiated in Terms of their Phenotypes of Differentiation Antigens or their Functions"
T lineage stem cells differentiated and developed from lymphoid stem cells derived from h~ Jni~tir stem cells proliferate and mature through each stage of differentiation and maturation under the control of adhesion with the thymic interstitial cells and of f~nrln~nous humoral factors.
Each stage of differentiation and maturation can be .

~ ~U9DS77 distinguished and classified by the phenotypes based on difference of kinds of differentiation antigens expressed on the cell surface, or by biological functions such as abilities of antigen specific proliferation, s cell activation, cytotoxicity and immunosuppression.
By applying the method of the present invention, it is possible to realize and achieve all stages of differentiation and maturation in vi~rQ using the "T lineage precursor cells" defined in a). The term matured T cells 10 dif Eerentiated in terms of their phenotypes of differentiation antigens or their functions" means T lineage cells in said any stage of differentiation and maturation.
Brief Deficription of Drawinqs Fig. 1 is a graph showing a correlation between the number of recovered viable cells and the dissolved oxygen concentration in a nutrient medium under each culture condition i Figs. 2 to 7 are graphs showing the distributions of the following varlous types of cells, which is dis-20 tinguished on the basis of cell sizes determined by using aflow cytometer, wherein relative complexities of the cells based on side-light scatter are plotted along the ordinates and relative cell sizes based on forward-light scatter are plotted along the abscissas.
That is:
Fig. 2 is a graph showing the cell distribution of mouse matured T cells, Fig. 3 is a graph showing the cell distribution of mouse fetal thymocytes immediately after these t~lymocytes are 3 o col 1 ec ted, Fig. 4 is a graph showing the cell distribution after mouse fetal thymuses are cultured by an afloat culture at a 20% oxygen concentration, Fig. 5 is a graph showing the cell distribution after mouse fetal thymuses are cultured by a submerged culture at a 596 oxygen concentration, .~

20~06~7 Fig. 6 is a graph showing the cell distribution after mouse fetal thymuses are cultured by a submerged culture at a 20% oxygen concentration, and Fig. 7 i8 a graph showing t~le cell distribution after mouse fetal thymuæes are cultured by a submerged culture at a 60% oxygen concentration;
Figs. 8 to 15 are graphs showing the cell distribu-tions showing the expression level of CD4 and/or CD8, each of which is a differentiation antigen on mouse T cell membranes.
lO These expression levels are distinguished by a fluorescence-linked antibody-mediated analysis using a flow cytometer, wherein the expression level of CD4 is plotted along the ordinates and the expression level of CD8 is plotted along the abscissas; that is:
Fig. 8 is a graph showing the cell distribution which represents the expression level of CD4 and/or CD8 of mouse matured T cells, Fig. 9 is a graph showing the cell distributio which represents the expression level of CD4 and/or CD8 of 20 mouse fetal thymocytes immediately after these thymocytes are collected, Fig. 10 i8 a graph showing the cell distribution which represents the expression level of CD4 and/or CD8 after the mouse fetal thymuses are cultured by an afloat culture at a 20% oxygen concentration, Fig. ll is a graph showing the cell distribution which represents the expression level of CD4 and/or CD8 after the mouse fetal thymuses are cultured by a submerged culture at a 5% oxygen concentration, Fig. 12 is a graph showing the cell distribution which represents the expression level of CD4 and/or CD8 after the mouse fetal thymuses are cultured by a submerged culture at a 2096 oxygen concentration, and Fig. 13 is a graph showing the cell distribution which represents the expression level of CD4 and/or CD8 after the mouse fetal thymuses are cultured by a submerged culture .
~.

~90~77 at a 40% oxygen concentration.
Fig. 14 is a graph 6ll0wing the cell distribution which represents the expreæ6ion level of CD4 and/or CD8 after the mou6e fetal thymuse6 are cultured by a 6ubmerged culture at a 60% oxygen concentration.
Fig. 15 is a graph showing the cell di6tribution which represents the expre66ion level of CD4 and/or CD8 af ter the mou6e fetal thymu6e6 are cultured by a 6ubmerged culture at an 80% oxygen concentration.
Fig. 16 i6 a graph 6howing the proliferation degree of ConA-reactive, functional T cell6 mea6ured by u6ing a tritium-labeled tllymidine ( 3H-TdR) incorporation te6t, w~lerein the cellular incorporation amount6 (cpm) of 3H-TdR a6 an index of DNA 6ynthe6i6 accompanying in T cell proliferation are plotted along the ordinate, and the dilution rate6 of the number of cell6 per a cell population of thymu6 after the culture are plotted along the absci6sa;
Fig. 17 i6 a graph 6howing the proliferatrion degree of allo-reactive, functional T cell6 mea6ured by u6ing a 2 0 tritium-labeled thymidine ( 3H-TdR ) incorporation te6t, wherein the cellular incorporation amounts (cpm) of 3H-TdR as an index of DNA synthesis accompanying in T cell proliferation are plotted along the ordinate, and the dilution rates of the number of cells per a cell population of thymus after the culture are plotted along the abscissa;
Fig. 18 is a graph showing the cell distribution which represents t~le expre66ion level of CD4 and/or CD8 after mouse thymic interstitial cells not containing mouse fetal t~lymocyte6 i6 cultured by a 6ubmerged culture at a 60% oxygen 30 concentration.
Fig. 19 i6 a graph 6howing the cell di6tribution which repre6ent6 the expression level of CD4 and/or CD8 af ter individually collected mouse thymic inter6titial cell6 and 300 mou6e fetal thymocyte6 are cultured by a 6ubmerged culture at a 60% oxygen concentration.
Fig. 20 is a graph showing the cell distribution ll ~ 2~0677 which represents the expression level of CD4 and/or CD8 of mouse matured T cells.
Fig. 21 is a graph showing a correlation between the oxygen concentration in the gas phase contacting with the nutrient medium and the dissolved oxygen concentration in tlle nutrient medium.
Best Mode of Carrying out the Invention The ~ ~nrl; ~ - lts of the present invention are described in detail by way of its examples. The present 10 invention is not limited to the following examples, as a matter of course.
Example 1 <1-1> Collection of Mouse Thymus Thymuses were aseptically collected by an ordinary method from 6-week old C57sL/6 mice (B6 young mice) and a 15-day fetuses of B6 mice (available from Nippon SLC Corp. ) obtained from time-mated C57BL/6 mice (B6 pregnant mice).
<1-2> Collection of Mouse Thymocytes and Counting The thymuses of the young mice and the mouse fetal 20 thymuses (FT), which were collected in 1-1, were gently minced with stainless steel mesh to afford young mouse thymocytes and mouse fetal thymocytes.
These thymocytes were suspended in an RPMI-1640 complete medium (available from Gibco Corp. ), and the numbers of viable cells present in the suspension were counted by a trypan blue dye exclusion test.
<1-3> Culture of T precursor cells at various Oxygen Concen-trations The fetal thymocytes (7.0 x 104 cells/lobe) obtained 30 from the 15-day fetuses of B6 mice were added to an RPMI-1640 complete medium containing sodium pyruvate, sodium hydrogen-carbonate, non-essential amino acids (available from Gibco Corp. ), 2-mercaptoethanol, penicillin, streptomycin, and 10%
fetal calf serum (FCS) in a 24-well microtlter plate. The plate was sealed in a plastic vessel (Gaspack (trademark) available from BBL Corp or Tedler (trademark) bag available ~2 .~

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~ 2~90~77 from SeikagakU Kogyo Inc. ), and air in the vessel was substituted with gas mixtures (available from Iwatani Sangyo Inc. ) respectively containing 5% carbon dioxide and 5~ oxygen, 5~ carbon dioxide and 20~ oxygen, 5~i carbon dioxide and 30%
oxygen, 5% carbon dioxide and 40% oxygen, 5% carbon dioxide and 60% oxygen, and 5% carbon dioxide and 80% oxygen. The cells were cultured by a submerged culture at 37C for 5 days.
In order to compare the present invention with the prior art, fetal thymocytes (7.0 x 104 cells/lobe) obtained 10 from 15-day fetuses of B6 mice were cultured by an afloat culture under the condition of at a carbon dioxide concentration of 5%, an oxygen concentration of 20% and 37C
for 5 days on a filter (diameter: 13 mm; pore size: 8 ~Lm available from Nucleopore Corp. ) floating in an RPMI-1640 complete medium containing sodium pyruvate, sodium hydrogen-carbonate, non-essential amino acids (available from Gibco Corp. ), 2-mercaptoethanol, penicillin, streptomycin, and a 10%
fetal calf serum (FCS) in a 24-well microtiter plate.
After the culture, the numbers of cells were counted 20 by the trypan-blue dye exclusion test as in 1-2 above.
Results are summarized in Table 1. In order to examine the relationship between the numbers of recovered viable cells of each population of cells after the culture and the dissolved oxygen concentration in the nutrient medium, the dissolved oxygen concentrations in the medium after completion of the culture were measured by a blood gas analyzer (stat Profile available from siomedical Corp. ), and results are shown in Fig. ~.

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,, ~ ~090~77 The cell6 proliferated up to about 33.2 x 104 cell6/lobe on the fift~l day by the conventional afloat culture, namely, 5-times cell proliferation was observed in comparison with the state prior to the culture.
On the other hand, in the submerged culture, the cell proliferation only up to about 2.4 x 104 cells/lobe and 3.7 x 104 cells/lobe were observed at oxygen concentrations of 5% and 20%, respectively. In contrast, the cells significantly proliferated at an oxygen concentration of 4096 or more. At oxygen concentrations of 40%, 60% and 80%
respectively, proliferations up to about 9.3 x 104 cells/lobe, 23.5 x 104 cells/lobe, and 28.8 x4 10 cells/lobe were observed. In particular, the cells multiplied about 4 times in comparison with the state prior to the culture at the oxygen concentration of 80%. The cell proliferation at the oxygen concentration of 80% was almost equal to that of the conventional cumbersome afloat culture which was not suitable for mass production due to the complexity of the process.
Example 2 Differentiation of T precursor cells (Expresæion of Dif f erentiation Antigens ) Following the same procedures as in 1-3 above, air available in a vessel was substituted with gas mixtures (from Iwatani Sangyo Inc. ) containing 5% carbon dioxide and 5%
oxygen, 5~ carbon dioxide and 20~ oxygen, 5% carbon dioxide and 40~ oxygen, 59~ carbon dioxide and 60% oxygen, and 5~
carbon dioxide and 80% oxygen. Then, cells were cultured in the vessel using a submerged culture and differentiation states of the grown cells were examined.
In order to compare the present invention with the prior art, cells were cultured by an afloat culture following the same pro~edures as in 1-3 above to examine the di f f erentiation states .
Mouse matured T cells were used as a control.
<2-1> Distribution of Cells The distribution of cells was measured by a flow .~--, ~ 20~067~
cytometer (FACScan (trademark) available from Becton Dickinson Corp. ) to observe the differentiation states of the cultured T precursor cells.
Results are shown in Figs. 2 to 7.
In these experiments, the distribution of cells roughly classified into blastic lymphoid cells, whose differentiation and maturation have not been completed, and small lymphoid cells whose differentiation and maturation have been completed and have a diameter of about 5 to 10 ~Im is 10 examined. By this examination of the cell distribution, it is able to observe the state of the differentiation and maturation of the T precursor cells.
Most of the cells in the population of mouse matured T cells (Fig. 2) were observed as small lymphoid cells.
Similarly, in a population of cells (Fig. 4) obtained by culturing mouse fetal t~lymuses by an afloat culture at an oxygen concentration of 20%, most of the cells were observed as small lymphoid cells.
On the other hand, in populations of cells (Figs.
20 5 and 6) obtained by culturing mouse fetal thymuses by a submerged culture at oxygen concentrations of 5% and 20%
respectively, most of the cells were observed as blastic lymphoid cells similar to the c~se of a population of t~le mouse fetal thymocytes (Fig. 3) immediately after being collected. The result showed that the differentiation and maturation were not achieved.
On the contrary to those results, in a population of cells (Fig. 7) cultured by a submerged culture at an oxygen concentration of 60%, most of cells were observed as small 30 lymphoid cells similar to the cases of a population of mouse matured T cells (Fig. 2) and the population of cells (Fig. ~) cultured by an af loat culture at an oxygen concentration of 20/. The result showed that t~le differentiation and maturation of the T precursor cells were achieved.
<2-2> Expre~sion of Differentiation Antigens ~ 2~9~7 Expreæsion level o~ mouse differentiation antigens CD4 and CD8 were measured by an ordinary method using a flow cytometer (FACBcan (trandemark) available from Becton Dickin60n corp. ) in accordance with a fluorescence-linked antibody-mediated analysis using monoclonal antibodies for each differentiation antigen of the mouse T cells, in order to observe the differentiation level of the T precursor cells af ter the culture .
The monoclonal antibodies used in this experiment o were a phycoerythrin (PE)-labeled anti-mouse CD4 monoclonal antibody (rat hybridoma GK1.5 available from Becton Dickinson Corp. ) and a fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 monoclonal antibody (rat hybridoma 53.6.7 available f rom Becton Dicl~inson Corp .
Results are shown in Table 2 and Figs. 8 to 15.

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Most of cells in the population of mouse matured T
cells (Fig. 8) were found to be CD4+ and/or CD8+. Further, most of these cell6 were CD4+ CD8+. Similarly, in a population of cells (Fig . 10 ) obtained by culturing mouse fetal thymocytes by an afloat culture at an oxygen concentration of 20%, 90% or more cells of total cells were found to be CD4+
andtor CD8+.
On the other hand, in populations of cells (Figs.
11 and 12) obtained by culturing mouse fetal thymocytes by a 10 submerged culture at oxygen concentrations of 5% and 20%
respectively, most of tlle cells were CD4 CD8 . The cells were, therefore, kept undifferentiated in terms of phenotypes of the differentiation antigens similar to the case of a population of mouse fetal thymocytes (Fig. 9) immediately after being collected.
In contra6t to these populations of cells, in a population of cells (Fig. 13) cultured by a submerged culture at an oxygen concentration of 40%, 80% or more of the total cells were CD4+ and/or CD8+ . In population6 of cells ( Figs .
20 14 and 15) cultured by a submerged culture at oxygen concentrations of 60% and 80% respectively, 90% or more of the total cells were CD4+ and/or CD8+ similar to the cases of t~le populations of cells (Fig. 8) obtained by culturing the popu-lations of mou6e matured T cells (Fig. 8) and the populations of cells (Fig. 10) cultured by an afloat culture at an oxygen concentration of 2096. Moreover, most of the cells in the populations of cells in Fig. 13 were CD4+ CD8+. The reæults showed that the differentiation and maturation of the T
precursor cells in terms of phenotypes oE the differentiation 30 antigenæ were achieved.
In addition, the ratio of cells expressing the differentiation antigens in the population of blastic lymphoid cells were increased as the oxygen concentration was raised in the submerged culture. In the population of blastic lymphoid cells cultured by submerged culture at oxygen concentrations of 6096 anù 80%, 70 to 80% of the total cell~

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expressed tlle differentiation antigen6.
The above results were similar to the ratio of cells expressing differentiation antigens in the population of blastic lymphoid cells obtained by performing an afloat culture at an oxygen concentration of 20%, indicating t~lat the differentiation in terms of phenotypes of the differentiation antigens was almost perfectly achieved.
<2-3~ Expression of T cell Receptor At one stage of differentiation and maturation, the 10 T precursor cells express an antigen receptor (T cell receptor; TCR) on the cell membrane. The TcR consists of subunits called ~-, ,3-, y- and o-chains. The TcR associated with CD3, which is one differentiation antigen serving as a molecule for intracellular signal transduction, thereby forming CD3-TcR. Therefore, by ~ m;ning the expressions of TcR~ and TcRyo and the expression of CD3, the differentiation level and the degree of the functional maturation can be evaluated. T cells constituting a mechanism of recognition of foreign antigens in vivo are mainly the cells expressing 2 0 TcR~ . The t~lymus produces a larger number of TcR~-expressing T cells than of TcRyo-expressing T cells and supply the periphery with the TcRc~-expressing T cells.
In this experiment, the expression levels of CD3, TcRo~ and TcRyo were examined following the same procedures as in 2-2 above by using monoclonal antibodies for their differentiation antigens; CD3, TcRo~ and TcRyo respectively.
The monoclonal antibodies used in this experiment were an anti-mouse CD3 monoclonal antibody (hamster hybridoma 145-2Cll available from Seikagaku Kogyo Inc. ), an anti-mouse 30 TcR~ monoclonal antibody (hamster hybridoma H57-597 ), and an anti-mouse TcRyo monoclonal antibody (hamster hybridoma 3A10).
A fluoresceinlabeled rabbit anti-hamster immunoglobulin (available from Caltag Corp. ) was used as a secondary antibody .
Results are shown in Table 3.

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~ 2,~0~7 As for the expression of CD3, ratio of CD3-positive cells was 63 . 2% in a population of cellæ cultured by a submerged culture at an oxygen concentration of 60%. This expre6sion ratio was equal to or higher than t~le expression ratio of 55.7% in the population of cells cultured by an afloat culture at an oxygen concentration of 20%, thus indicating that the differentiation of the T precursor cells was achieved.
The expressions of TcR~,~ and TcRyo occurred as 10 follows. The TcRa~-expressing cells and TcRyo-expressing cells were respectively 48.8% and 4.7% in the population of cells cultured by an afloat culture at an oxygen concentration of 20%. That is, the differentiation of the TcRx,~-expressing T
cells occurred selectively.
On the other hand, the TcR~,~-expressing cells were respectively 11.3% and 19.396 in the population of cells cultured by a submerged culture at oxygen concentrations of 596 and 20%, while the TcRyo-expressing cells were 28.5% and 24.3% in these populations of cells. The differentiation to 20 TcRyo-expressing T cells mainly occurred, and the differen-tiation to the TcR~13-expressing ~ cells was insufficient.
In contrast to these results, tlle TcRo~,~-expressing cells and TcRyo-expressing cells were respectively 46 . 9% and 14 . 4% in a population of cells cultured by a submerged culture at an oxygen concentration of 60%. The differentiation to the TcR~,~-expressing T cells selectively occurred equally to that of the population of cells cultured by the afloat culture at the oxygen concentration of 20%, and indicating that normal differentiation of the T precursor cells was achieved.
In addition, the numbers of the recovered cells expres6ing CD3, TcR~ and TcRy~ respectively, which were generated by the culture under the respective culture conditions were counted. CD3-, TcRG~-, and TcRy~-expressing cells were not sufficiently obser~Ted in populations of cells cultured by a submerged culture at oxygen concentrations of 5% and 20% respectively. However, CD3-positive and TcR~-.~ , ~ .

~ 20~06~7 positive cells were generated by 7 to 13 times and by 14 to 39, times respectively, in a population of cells cultured by a submerged culture at an oxygen concentration of 60%. This result is almost equal to the result in the case of the population of cells cultured by the afloat culture at the oxygen concentration of 2096.
Example 3 Generation of Functional T cells.
<3-1> Proliferation Activity of Functional T cells Induced with Mitogen Substances which activate lymphocytes and induce DNA
synthesis and cell prollferation are generically called mitogens (division accelerating factor). Concanavalin-A
(ConA), one of the mitogens, acts through a CD3-TcR (shown in 2-3 ) complex which participates in antigen recognition on the T cells, thereby inducing the proliferation of T cells. The proliferation of the T cells induced by ConA is maintained by cytokines such as Interleukin-2 ( IL-2 ) which is secreted into the culture supernatant from the T cells upon ConA stimulus.
In this experiment, following the same procedures as in 2-3 described above, thymuses were cultured for s days at various oxygen concentrations, using a 24-well microtiter plate. Thus obtained T cells were cultured in an RPMI-1640 complete medium containing a 10~ FCS in the presence of ConA
(15 llg/mQ; available from ~Tector Corp.) and IL-2 (50 units;
available from Genzyme Corp. ) using a 96-well microtiter plate, and then, derivation and generation of functionally matured T cells having ConA reactivity were examined.
The ConA reactivity of the T cells was determined 30 as follows. The cultures were performed for 3 days, then, 1.0 uCi of tritium-labeled thymidine (3E~-TdR) was added to each medium at 6 hours before from the end of the culture, and cells were harvested onto glass filters. Then, a scintillator (Clear-SolI available from Nakarai Techs Inc. ) was added to each sample, and intracellular incorporation of the 3H-TdR was measured using a liquid scintillation counter.

.
~i ~ 209~7~
Results are s~lowh in Fig. 16.
The proliferation of populations of cells cultured by a submerged culture at oxygen concentrations 5% and 20%
were 1/4 to 1/3 respectively in comparison with that of a population of cells cultured by an afloat culture at an oxygen concentration of 20%. On the contary, the proliferation of a population of cells cultured by a submerged culture at an oxygen concentration of 60% was almost equal to tilat in the case of a population of cells cultured by an afloat culture 10 at an oxygen concentration of 20%, showing that the functionally matured T cells having the ConA reactivity were derived and generated at the same degree with the di~ferentiation in the thymuses of young mice and the differentiation in the fetal thymuses by the afloat culture.
<3-2> Reactivity of Functional T cells against Allo (BALB/C
Nouse) Spleen Cells T cells are educated in the thymus and acquire reactivity against allo mou6e cells which are non-self, thereby causing graft rejection, for example. Similarly to 20 the action of ConA described in 3-1 above, this reaction is also caused via a TcR-CI)3 complex on the T cells which have matured in tlle thymus.
In this experiment, thymuses were cultured for 6 days at various oxygen concentrations, using 24-well microtiter plates as in the method in 2-3 above. T cells thus obtained were cultured in a 96-well microtiter plate (5 x 105 cells/well ), to which were added with cell suspensions which were obtained by pretreating spleen cells collected from 6-week old BALB/C mice with mitomycin C (25 llg/mQ, 3?C, 30 30 minutes), subsequently was~ing the heated cells with an RPMI-1640 medium three times. Then, generation of t~le T cells having reactivity against the allo cells was ~Amin~.
The allo reactivity of the T cells was determined as follows. The cultures were performed for 5 days, tilen, 1.0 ~Ci of tritium-labeled thymidine (3H-TdR) was added to e~ch medium at 6 hours before the end of the culture, and cells ~' ~ ~090677 were harvested onto gla6s filters. A scintillator (Clear-SolI
available from Nakarai Techs Inc. ) wa6 added to each sample, and intracellular incorporation of the 3H-TdR was measured using a liquid scintillation counter.
Results are shown in Fig. 17.
The proliferation of populations of cells cultured by a submerged culture at oxygen concentrations 5% and 20%
were 1/10 to 115 compared with that of a population of cells cultured by an afloat culture at an oxygen concentration of o 20~. In contrast to the results, the proliferation of a population of cells cultured by a submerged culture at an oxygen concentration of 60% was almost e;aual to that of a population of cells cultured by an afloat culture at an oxygen concentration of 20%, which indicated that the functionally matured T cells having the allo reactivity were derived and generated at the same degree with t}1e differentiation in the thymuses of young mice and the differentiation in the fetal thymuses by the af loat culture .
Example 4 Differentiation and Maturation of T precursor cells by Combination of T precursor cells and Interstitial Cells of Lymphoid Tissues.
Normal differentiation and maturation of T precursor cells using mouse fetal thymuses by applying the techni~ue of the present invention have been described in Examples 1 to 3.
According to the present invention, furthermore, normal differentiation and maturation of T precursor cells can be also achieved by using a combination of T precursor cells and interstitial cells of lymphoid tissues ( e . g ., thymic 30 epithelial cells, fibroblasts, other interstitial cells of lymphoid tissues, and/or intercellular substances ) surrounding the T precursor cells which are respectively collected from the same or different individuals of the same or different species. An example is described below.
Thymuses obtained from 15-day fetuses of B6 mice (available from Nippon SLC Corp. ) were cultured by an afloat . ~ , ...
. .

~" 209~677 culture in RPMI-1640 complete medi~lm containing 1. 35 mM
2'-deoxyguanosine (dGuO) and 10% ~etal calf serum (FCS) for 6 days, followed by further afloat culture for 12 hours in tlle complete medium without dGuO, thereby obtaining a thymic interstitial cells free from blood cells.
The thymic interstitial cells thus obtained were seeded in a 96-well round bottom plate, and RPMI-1640 complete medium was added to the plate (200 IlQ/well). Then, T precursor cells (mouse fetal thymocytes) obtained by the same procedures lO as in Example 1-2 were added at 30 cells/well or 300 cells/well. The plate was sealed in a plastic vessel (Gaspack (trademark) available from BBL Corp. ), and air in the vessel was substituted with gas mixture (available from Iwatani Sangyo Inc. ) containing 5% of carbon dioxide, 60% of oxygen, and 35% of nitrogen. Thereafter, the samples were cultured by a submerged culture for 7 days.
After the culture, the recovered cells and the thymic tissues were gently minced with a 6tainless steel mèsh to obtain T cells. In order to observe the differentiation and 20 maturation degree of T precursor cells after the culture, t~le expression level of CD4 and CD8, which are differentiation antigens of the mouse T cells was measured following the same procedures as in Example 2-2.
A culture obtained by culturing only thymic interstitial cells without adding the T precursor cells (mouse fetal thymocytes) by a submerged culture at an oxygen concentration of 6096 was used as 2 control, and mouse matured T cells were used for comparison.
Results are shown in Figs. 18 to 20.
Most of the cells in the population of mouse matured T cells group (Fig. 20) were CD4+ and/or CD8+. Further, most of these cells were CD4+ CD8+.
In the population of cells (Fig. 18) cultured by a submerged culture at an oxygen concentration of 60% without adding the T precursor cells (mouse fetal thymocytes), very few cells (including thymic epithelial cells, fibroblasts, and .i .

so on) were recovered. very few cells exhibited CD4+ and/or CD8+ .
On t~le other hand, in the populations of cells obtained f rom the culture by a combination of mouse thymic interstitial cells and T precursor cells (mouse fetal thymocytes) (30 or 300 cells each)~ which were individually collected, by a submerged culture at an oxygen concentration of 60%, a large number of cells were found to be CD4+ and/or CD8+. When 30 cells/well mouse fetal thymocytes were used, 30 10 to 40% cells exhiblted CD4+ and/or CD8+. When 300 cells/well of mouse fetal thymocytes were used (Fig. 19), 90~6 or more of the cells exhibited CD4+ CD8+.
The result in tlle culture by combination is equiva-lent to that in the case of the population of mouse matured T cells, showing that the differentiation and maturation of the T precursor cells in terms of their phenotypes of the differentiation antigens were achieved even by a culture with a combination of the mouse fetal thymocytes and the mouse thymic interstitial cells which were individually collected.
20 Example 5 Culture of Allogenic T precursor cells in Matured T cells Deficient Animal Model.
When the thymus functions for proliferating, differentiating, and maturing B precursor cells and/or T
precursor cells are deficient or the matured B cells and/or the matured T cells are congenitally deficient, various diseases owing to a deficient immune system are induced. Even if the matured s cells or matured T cells were administered in an adoptive immunotherapy, matured B cells or matured T
30 cells having an appropriate antigen specificity cannot be induced due to the deficiency of lymphoid precursor cells (e.g., B precursor cells and T precursor) or the abnormality of the proliferation, differentiation, and maturation process in the thymus. Therefore, there is little significance of therapy. In order to examine the tllerapeutic significance value of the present invention in such a model, the following ~, ~, ~ 20~0~7 experiment was performed.:
Thymuses we~e aseptically collected from 6-week old SCID mice (severe combined immunodeficient mouse; available from Fox Chase Corp. ) wllich are the model of mice having defects in abilities of differentiation and maturation of B
precursor cells and T precursor cells, following the same procedures as in Example 1-1. The obtained each thymus was divided into four pieces. Eac~l piece was added to an RPMI-1640 complete medium using a 96-well microtiter plate.
Fetal thymocytes as T precursor cells were obtained from 14-day fetuses of C57BL/6 mice (available from Nippon SLC
Inc. ) following the same procedures as in Examples 1-1 and 1-2 and they were added by the number of 5 x 104 cells/well into t~le Rl?MI-1640 complete medium containing the thymus pieces of the SCID mouse.
The cells in t~1e culture plate were sub; ected to a submerged culture at 37C for 7 days, in an atmosphere having a carbon dioxide concentration of 5% and an oxygen concentration of 20% (normal atmospheric condition) and in 20 an ai ~srh~re having a carbon dioxide concentration of 5% an oxygen concentration of 80%.
In order to improve efficiency of the differentia-tion and maturation of tile T precursor cells, a culture was performed under the same conditions using the SCID mouse thymuses which T precursor cells having no differentiation or maturation abilities were removed by radiation ( 2 . 5 Gr ) .
The number of cells after culture was counted by the trypan-blue dye exclusion test.
The results are shown in Table 4. Eac~l described 30 value is an average of experimental values obtained using four-split thymus pieces.

~, . ~
=

~90677 Table 4 Gas Phase Nulnber of cells after concentration culture (cells/lobe ( vol96 ) 2 N2 CO2 E~adiation- E~adiatioll-o Treated Non-Treated 7s 5 ] . 3 x 104 2 . 8 x 104 7s 5 1.6 x 104 s 3.8 x 104 In experiments using botil radiation-treated thymuses and nontreated thymuses, populations of cells cultured at an oxygen concentration of 80% exhibited twice or more of a survival rate of T precursor cells compared with that of populations of cells cultured at an oxygen concentration of 20~6 (normal atmospheric condition).
In comparison between the populations of cells cultured at an oxygen concentration of 80% using radiation-treated or nontreated thymuses, the population of cells using the radiation-treated thymuses exhibited a more significant survival rate of T precursor cells than the population of cells using the nontreated thymuses.
Differentiated and matured T cells were observed in the viable r .
cells. 2090677 Example 6 Culture of Human T Precursor Cells at High Oxygen Concentration .
A part of the thymus close to the heart of a 2-year old patient suffering from a heart disease was removed in a surgical operation. The obtained human thymus was divided into pieces each having a same weight (about 10 mg). The prepared thymus pieces (5 x 105 cells/thymus piece) were treated 10 according to the Game procedures as in Example 1, followed by a submerged culture at 37C for 14 days in an atmosphere having a carbon dioxide concentration of 5% and an oxygen concentration of 20%, as well as in an atmosphere having a carbon dioxide concentration of 5% and an oxygen concentration of 70%.
The numbers of cells after the culture were counted by the trypan blue dye exclusion method.
Results are 6ummarized in Table 5.
Table 5 I

Gas P~lase concentration (Vol%) Number of Cellsafter Culture (cells/lobe~

20 75 5 0.64 x 105 70 15 S l . 50 x l05 gimilar to the results of Example 1 using the mouse thymocytes, in this experiment using the human thymocytes, the survival rate of cells in the culture at the oxygen i "~, 2~ 7~
concentration of 70% (high oxygen concentration ) was found to be significantly twice or more than with that found at the oxygen concentration of 20% (normal atmospheric condition).
Judging from the results obtained in Examples 5 and 6, the pre6ent invention enabled derivation of normally matured T cells from the different strain or same strain T
precursor cells even in tissues such as human thymuses and matured T cell-deficient animal model such as SCID mice.
Therefore, it can be given as a conclusion that the present 10 invention is highly applicable to research and development of cell vaccines and therapeutic agents for various autoimmune diseases or immune diseases due to virus infections, such as AIDS (acquired immuno-deficiency syndrome).
Example 7 Correlation between Gas Ph~se Oxygen Concentration and Liquid-Phase Dissolved Oxygen Concentration.
The following experiment was conducted to examine the correlation between the oxygen concentration in the gas phase contacting a nutrient medium and the dissolved oxygen 20 concentration in the nutrient medium.
A 96-well microtiter plate containing an RPMI-1640 complete medium containing or not containing mouse fetal thymuses (7 x 104 cells/lobe) obtained in Example 1 was sealed in a plastic vessel (Gaspack (trademark) available from B3L
Corp. or Tedler (trademark) bag available from Seikagaku Kogyo Inc. ) . As shown in Fig. 21, the carbon dioxide concentration was maintained constant (5%), and gas mixture (available from Iwatalli Sangyo Inc. ) having a predetermined constant mixing ratio of t~le oxygen concentration ( % ) to the 30 nitrogen concentration (%) was blown from a vent of the ves~el to sufficiently substitute the air in the vessel with the gas mixture. Thereafter, the microtiter plate Was stood in the vessel at 37C for 1 to 5 days.
After the concentrations of carbon dioxide, oxygen, and nitrogen were in equilibrium between the gas phase and the liquid phase, the dissolved oxygen concentration (mmHg, uM) ~ 2090677 in the nutrient medium (liquid phase~ was measured using an automatic gas analyzer (available from NoYa Corp. ) .
Results are shown in Fig. 21.
When the volume of the gas mixture ( gas phase ) contacting with the nutrient medium was relatively larger enough than the volume of the nutrient medium (liquid phase), a proportional relationship was obtained between the gas phase oxygen concentration and the liquid phase dissolved oxygen concentration. In addition, when the volume of the gas phase 10 was not relatively large, it was f ound that a correlation falling within the region (a hatched portion in Fig. 21) below the proportional line can be established.
As has been in detail described above, according to the present invention, T lineage precursor cells are cultured in a nutrient medium under the condition that a dissolved oxygen concentration in the nutrient medium is higher than that in the nutrient medium under the normal atmospheric air, i . e ., under the condition that the dissolved oxygen concentration is raised by mean6 of raising the oxygen 2 0 concentration in the gas phase contacting the nutrient medium to 40 vol% through 95 vol96. By raising the oxygen concentration in the gas phase to 40 96vol - 9o %vol, the dissolved oxygen concentration in the nutrient medium can be raised to approximately 360 ~M- 855 ,~LM. sy applying the method of the invention, not only can T lineage cells be cultured in a large scale ~ vi~ by a ~ubmerged (suspension) culture as a general method of culturing cells or tissues without using special tools, but also it is possible to realize in Vit~Q the proliferation process of the T precursor 30 cells and normal differentiation and maturation processes of T precursor cells in terms of the phenotypes of the dif-ferentiation antigens and their functions. It is also possible, by a ~ d (suspension) culture without using special tools, to derive and generate matured T cells in terms of their phenotypes of differentiation antigens and their functions, which have appropriate antigen recognition ability.

~B
. . ., i .

,. 2~go677 Therefore, the present invention can contribute to investigate details of differentiation and maturation r-~h~n~ c:m of the T
lineage cells.
FurthG e, by using the method of the pre6ent invention, T lineage precursor cells are cultured in a nutrient medium containing an antigen which induces infectiou6 disea6es, such as a hepatitis virus or HIV (human immuno-deficiency virus), allergic diseases, or autoimmune diseases, thereby deriving T cells which recognize the 10 antigen . Theref ore, appropriate T-cell vaccines capable of suppressing or treating the diseases such as infectious disea6es, allergic diseases, and autoi ^ diseases induced by the antigen can be prepared.

fB
-

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for culturing T lineage precursor cells to produce matured T cells comprising a step of culturing T
precursor cells in a nutrient medium contacting a gas phase having an oxygen content ranging from 40% to 95%, thereby said nutrient medium having a dissolved oxygen concentration ranging from 360 µM to 855 µM, to obtain matured T cells.

2. A method according to claim 1, wherein the step of culturing T precursor cells consists of culturing tissue containing T precursor cells.

3. A method according to claim 1 or 2, wherein said oxygen content of the gas phase ranges from 60% to 95%.

4. A method according to claim 1 or 2, wherein said dissolved oxygen concentration ranges from 540 µM to 855 µm.

5. A method according to claim 1, wherein said T
precursor cells are obtained from tissue selected from the group consisting of thymus, bone marrow and liver.

6. A method according to claim 2, wherein said tissue containing T precursor cells is obtained from an organ selected from the group consisting of thymus, bone marrow and liver.

7. A method according to claim 1 or 2, wherein said nutrient medium contains at least one antigen which induces infectious diseases, allergic diseases or autoimmune diseases.

8. A method according to claim 1 or 2, which further comprises collecting the matured T cells so obtained.

9. A method for culturing T lineage precursor cells to produce matured T cells comprising a step of culturing T
precursor cells together with interstitial cells of a lymphoid tissue in a nutrient medium contacting a gas phase having an oxygen content ranging from 40% to 95%, thereby said nutrient medium having a dissolved oxygen concentration ranging from 360 µM to 855 µM, to obtain matured T cells.

10. A method according to claim 9, wherein the step of culturing T precursor cells consists of culturing tissue containing T precursor cells.

11. A method according to claim 9 or 10, wherein said oxygen content of the gas phase ranges from 60% to 95%.

12. A method according to claim 9 or 10, wherein said dissolved oxygen concentration ranges from 540 µM to 855 µM.

13. A method according to claim 9, wherein said T
precursor cells are obtained from tissue selected from the group consisting of thymus, bone marrow and liver.

14. A method according to claim 10, wherein said tissue containing T precursor cells is obtained from an organ selected from the group consisting of thymus, bone marrow and liver.

15. A method according to claim 9, wherein said interstitial cells of lymphoid tissue and said T precursor cells are respectively obtained from a single individual.

16. A method according to claim 10, wherein said interstitial cells of lymphoid tissue and said tissue containing T precursor cells are respectively obtained from a single individual.

17. A method according to claim 9, wherein said interstitial cells of lymphoid tissue and said T precursor cells are respectively obtained from different individuals of the same species.

18. A method according to claim 10, wherein said interstitial cells of lymphoid tissue, and said tissue containing T precursor cells are respectively obtained from different individuals of the same species.

19. A method according to claim 9, wherein said interstitial cells of lymphoid tissue and said T precursor cells are respectively obtained from individuals of different species.

20. A method according to claim 10, wherein said interstitial cells of lymphoid tissue and said tissue containing T precursor cells are respectively obtained from individuals of different species.

21. A method according to claim 9 or 10, wherein said nutrient medium contains at least one antigen which induces infectious diseases, allergic diseases or autoimmune diseases.

22. A method of claim 9 or 10, which further comprises collecting the matured T cells so obtained.